Decoding Of Messenger RNA Research Articles

Structures of the ribosome bound to EF-Tu-isoleucine tRNA elucidate the mechanism of AUG avoidance.

The frequency of errors upon decoding of messenger RNA by the bacterial ribosome is low, with one misreading event per 1 × 104 codons. In the universal genetic code, the AUN codon box specifies two amino acids, isoleucine and methionine. In bacteria and archaea, decoding specificity of the AUA and AUG codons relies on the wobble avoidance strategy that requires modification of C34 in the anticodon loop of isoleucine transfer RNAIleCAU (tRNAIleCAU). Bacterial tRNAIleCAU with 2-lysylcytidine (lysidine) at the wobble position deciphers AUA while avoiding AUG. Here we report cryo-electron microscopy structures of the Escherichia coli 70S ribosome complexed with elongation factor thermo unstable (EF-Tu) and isoleucine-tRNAIleLAU in the process of decoding AUA and AUG. Lysidine in tRNAIleLAU excludes AUG by promoting the formation of an unusual Hoogsteen purine-pyrimidine nucleobase geometry at the third position of the codon, weakening the interactions with the mRNA and destabilizing the EF-Tu ternary complex. Our findings elucidate the molecular mechanism by which tRNAIleLAU specifically decodes AUA over AUG.

Premature aging in mice with error-prone protein synthesis.

The main source of error in gene expression is messenger RNA decoding by the ribosome. Translational accuracy has been suggested on a purely correlative basis to positively coincide with maximum possible life span among different rodent species, but causal evidence that translation errors accelerate aging in vivo and limit life span is lacking. We have now addressed this question experimentally by creating heterozygous knock-in mice that express the ribosomal ambiguity mutation RPS9 D95N, resulting in genome-wide error-prone translation. Here, we show that Rps9 D95N knock-in mice exhibit reduced life span and a premature onset of numerous aging-related phenotypes, such as reduced weight, chest deformation, hunchback posture, poor fur condition, and urinary syndrome, together with lymphopenia, increased levels of reactive oxygen species–inflicted damage, accelerated age-related changes in DNA methylation, and telomere attrition. Our results provide an experimental link between translational accuracy, life span, and aging-related phenotypes in mammals.

Mechanistic Insight into the Reactivation of BCAII Enzyme from Denatured and Molten Globule States by Eukaryotic Ribosomes and Domain V rRNAs.

In all life forms, decoding of messenger-RNA into polypeptide chain is accomplished by the ribosome. Several protein chaperones are known to bind at the exit of ribosomal tunnel to ensure proper folding of the nascent chain by inhibiting their premature folding in the densely crowded environment of the cell. However, accumulating evidence suggests that ribosome may play a chaperone role in protein folding events in vitro. Ribosome-mediated folding of denatured proteins by prokaryotic ribosomes has been studied extensively. The RNA-assisted chaperone activity of the prokaryotic ribosome has been attributed to the domain V, a span of 23S rRNA at the intersubunit side of the large subunit encompassing the Peptidyl Transferase Centre. Evidently, this functional property of ribosome is unrelated to the nascent chain protein folding at the exit of the ribosomal tunnel. Here, we seek to scrutinize whether this unique function is conserved in a primitive kinetoplastid group of eukaryotic species Leishmania donovani where the ribosome structure possesses distinct additional features and appears markedly different compared to other higher eukaryotic ribosomes. Bovine Carbonic Anhydrase II (BCAII) enzyme was considered as the model protein. Our results manifest that domain V of the large subunit rRNA of Leishmania ribosomes preserves chaperone activity suggesting that ribosome-mediated protein folding is, indeed, a conserved phenomenon. Further, we aimed to investigate the mechanism underpinning the ribosome-assisted protein reactivation process. Interestingly, the surface plasmon resonance binding analyses exhibit that rRNA guides productive folding by directly interacting with molten globule-like states of the protein. In contrast, native protein shows no notable affinity to the rRNA. Thus, our study not only confirms conserved, RNA-mediated chaperoning role of ribosome but also provides crucial insight into the mechanism of the process.

Novel base-pairing interactions at the tRNA wobble position crucial for accurate reading of the genetic code.

Posttranscriptional modifications at the wobble position of transfer RNAs play a substantial role in deciphering the degenerate genetic code on the ribosome. The number and variety of modifications suggest different mechanisms of action during messenger RNA decoding, of which only a few were described so far. Here, on the basis of several 70S ribosome complex X-ray structures, we demonstrate how Escherichia coli tRNALysUUU with hypermodified 5-methylaminomethyl-2-thiouridine (mnm5s2U) at the wobble position discriminates between cognate codons AAA and AAG, and near-cognate stop codon UAA or isoleucine codon AUA, with which it forms pyrimidine–pyrimidine mismatches. We show that mnm5s2U forms an unusual pair with guanosine at the wobble position that expands general knowledge on the degeneracy of the genetic code and specifies a powerful role of tRNA modifications in translation. Our models consolidate the translational fidelity mechanism proposed previously where the steric complementarity and shape acceptance dominate the decoding mechanism.

Structural basis for selectivity and toxicity of ribosomal antibiotics.

Ribosomal antibiotics must discriminate between bacterial and eukaryotic ribosomes to various extents. Despite major differences in bacterial and eukaryotic ribosome structure, a single nucleotide or amino acid determines the selectivity of drugs affecting protein synthesis. Analysis of resistance mutations in bacteria allows the prediction of whether cytoplasmic or mitochondrial ribosomes in eukaryotic cells will be sensitive to the drug. This has important implications for drug specificity and toxicity. Together with recent data on the structure of ribosomal subunits these data provide the basis for development of new ribosomal antibiotics by rationale drug design.

A conformational switch in Escherichia coli 16S ribosomal RNA during decoding of messenger RNA.

Direct evidence is presented for a conformational switch in 16S ribosomal RNA (rRNA) that affects tRNA binding to the ribosome and decoding of messenger RNA (mRNA). These data support the hypothesis that dynamic changes in rRNA structure occur during translation. The switch involves two alternating base-paired arrangements apparently facilitated by ribosomal proteins S5 and S12, and produces significant changes in the rRNA structure. Chemical probing shows reciprocal enhancements or protections at sites in 16S rRNA that are at or very near sites that were previously crosslinked to mRNA. These data indicate that the switch affects codon-anticodon arrangement and proper selection of tRNA at the ribosomal A site, and that the switch is a fundamental mechanism in all ribosomes.

Messenger RNA translation in prokaryotes: GTPase centers associated with translational factors

During the decoding of messenger RNA, each step of the translational cycle requires the intervention of protein factors and the hydrolysis of one or more GTP molecule(s). Of the prokaryotic translational factors, IF2, EF-Tu, SELB, EF-G and RF3 are GTP-binding proteins. In this review we summarize the latest findings on the structures and the roles of these GTPases in the translational process.

Photochemical modification of transfer RNA and its effect on aminoacyl RNA synthesis

Transfer RNA was illuminated with visible light in the presence of methylene blue or the carcinogen 4-nitroquinoune- N-oxide. This procedure caused the loss of ultraviolet absorption of the guanine moiety. Analysis of base composition showed a decrease in guanine content, but no change in the other base components. The modification proceeded more rapidly in water than in buffer solution. The modification of polynucleotides proceeded slower than that of oligo- or mononucleotides. A scission of the backbone of the polynucleotide chain did not occur by this modification, but the configuration of tRNA was greatly altered. The guanine-modified tRNA was resistant to RNase T 1 digestion, whereas it was digested by pancreatic RNase IA, RNase T 2 or by alkali, as for the case of native tRNA. Amino acid-accepting ability of tRNA was destroyed with time of illumination. The inactivation rate varied for each amino acid. A significant decrease in the activity of proline acceptance was observed. This result supports the hypothesis that the anticodon sequence in tRNA would function in the recognition of an aminoacyl-RNA synthetase as well as in the decoding of messenger RNA.